Effects of Some Fatty Acid Esters on the Viability and Transplantability of Ehrlich Ascites Tumor Cells

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[CANCER RESEARCH 31, 501-504, May 1971] Effects of Some Fatty Acid Esters on the Viability and Transplantability of Ehrlich Ascites Tumor Cells

Akiko Kato, Kunio Ando, Gakuzo Jamura, and Kei Arima Laboratory of Microbiology, Department of Agricultural Chemistry, University of Tokyo, Tokyo, Japan

SUMMARY at higher pH values, only 2-decenoic, linoleic, and linolenic acids showed the activity. Fatty acids, monoglycerides, and some esters of fatty acids Unsaturated fatty acid fraction from the liver of show antitumor activity against Ehrlich ascites tumor in mice. X-ray-irradiated rabbit has been reported to have in vivo For investigation of the in vivo mode of action of these lipids, antitumor activity against the Brown-Pearce sarcoma of rabbit, the effects of those fatty acid esters on animal cells in vitro Ehrlich ascites tumor in solid form, and several human were studied in sheep red blood cells and Ehrlich ascites tumor carcinomas. The study on the chemical analysis of the fatty cells. Throughout these studies, we used fatty acid sucrose acid fraction was carried out by Seno and Yamamota (16), and esters because they are soluble in water, so that we could they found that the main components were hexadecanoic, obtain reproducible results without interference from the octadecanoic, octadecenoic, and octadecadienoic acids. solubility of the samples. Through the in vivo experiments with Ehrlich ascites tumor Most of the fatty acids and their esters showed hemolytic in mice, we studied the antitumor activity of each fatty acid action in sheep red blood cells and tumor cell-killing activity in (1), each monoglyceride (7), and other fatty acid esters (8). vitro. The tumor cells treated with such agents in vitro lost Our results indicated that the antitumor activity of the fatty their transplantability in mice. The sucrose monoester of lauric acids depended upon the number of the carbon chain and acid, which showed the strongest in vivo antitumor activity, the degree and the position of unsaturation. It was also found exhibited a strong in vitro activity, but polyoxyethylene that some esters were active while others were not. In the sorbitan monolaurate (Tween 20), which had no antitumor course of these studies, we realized that the insolubility of the activity, showed the stronger hemolytic and tumor cell-killing fatty acids and their esters in water might affect the results, or activities. These results indicate that antitumor activity of at least it was a problem to solve in carrying out the some fatty acid esters cannot be explained only by their experiments. Tolnai and Morgan (20), meeting with the same physical attack of the tumor cells. problem, studied the amino acid-fatty acid salts, which were water soluble, and found that L-lysine and L-arginine laurate were effective against the Ehrlich and TN3 ascites tumor cells INTRODUCTION in vitro. We examined the sucrose ester of fatty acids that were freely soluble in water. In our screening for antitumor antibiotics, we found that The antitumor activity of sucrose esters of 7 fatty acids was fatty acids and monoglycerides isolated from acetone extracts studied and reported in a previous paper (8). As summarized in of some fungal mycelia show antitumor activity in vivo against Table 1, all the sucrose esters inhibit the tumor growth. This Ehrlich ascites tumor in mice (3, 5). Chemical study of the finding of tumor inhibition by fatty acid sucrose esters fatty acids and the monoglycerides indicated that they are the enabled us to study the mechanism of action of fatty acids and mixture of the fatty acids or monoglycerides of fatty acids their esters on tumor cells. with carbon numbers of 16 and 18 (2, 6). Most of the works on the activity of fatty acids and their Antitumor activity of fatty acids was first reported by esters thus far reported were done with water-insoluble Nakahara (10-13). He reported that olive oil and fatty acids materials. All the works by Townsend et al. (21, 22), Morgan were effective in increasing the resistance of mice to several et al. (9), and Tolnai et al. (17-19) were carried out with forms of transplantable tumors. water-insoluble fatty acids themselves, as was the work of Townsend et al. (21,22) found that lO-hydroxy-2-decenoic Bennett et al. (4), who studied the effect of fatty acids on acid from royal jelly suppressed the development of a plasma membrane of Ehrlich ascites tumor cells and mouse transplantable mouse leukemia and the formation of ascitic erythrocytes. tumors in mice. They successively investigated the in vitro antitumor activity of mono- and dicarboxylic acids and their In these experiments, we used sucrose esters of fatty acids as in vivo antitumor-active materials, and Tweens as fatty acid hydroxy- and keto- derivatives (9, 17-20), and it was found that most of the mono- and dicarboxylic acids, when mixed derivatives with water solubility, which are inactive against the tumor in vivo. Hemolytic activity and in vitro attack of the with 3 different ascites tumor cells at pH values below 5.0 and tumor cells were studied, and results were compared with in prior to inoculation into mice, completely suppressed the vivo activity. In the study of the in vitro attack of the tumor development of the ascites tumor but when acids were mixed cells, we utilized a differential staining method for dead and Received February 2, 1970; accepted December 21, 1970. live cells and transplantability assay.

MAY 1971 501

Table I Antitumor activity of some esters of Â¡auricacidagainst Ehrlich ascites tumor in mice About 1 million of the tumor cells were implanted i.p. into 5-week-old ddY mice. A solution or suspension of the samples in 0.86% NaCl solution was administered once daily for 5 successive days. Six mice were used for each group. Control mice were given injections of 0.2 ml of the NaCl solution. 7daysBody weight time CompoundsMonolaurinMethyl Tumor(g)+1.3+4.0+2.0+2.9++ gain (days)>2926>272417182221>30>30>3026>3026252816

laurateSorbitan

laurateTween +3.7+++ +4.2+++ 20Sucrose +6.6+++ +6.9+2.9+ laurateSucrose 1.4+1.8+3.0+1.5+2.9+1.4+1.2+++ myristateSucrose

palmitateSucrose

linoleateControlDose(mg/mouse/day)10.02.520.05.010.05.010.02.516.04.020.05.010.02.518.04.5After

+8.4Survival

Hemolytic Activity MATERIALS AND METHODS 012345678 -Iâ€”Iâ€”Iâ€”Iâ€”iâ€”\â€”I 1â€”f- Sucrose Monoesters of Fatty Acids. These were synthesized Agents with strong antitumor activity according to the method of Osipow et al. (15). About 3 moles Ci2-sucrose of sucrose are dissolved in dimethylformamide, and 1 mole of Cm-sucrose methyl ester of fatty acid is added with 0.1 mole of potassium Cie-sucrose C|'e-sucrose carbonate as an alkaline catalyst. The reaction mixture is maintained at 90-95Â° under 80 to 100 mm Hg pressure. After Cfe-sucrose C,4-PG 6 to 9 hr, the solvent is distilled off and the residue is dried in a vacuum to obtain sucrose ester, monoester being the main Microbio! MG component. Other Esters of Fatty Acids. These were all available as Agents with weak antitumor activity Ciu-MG chemicals or industrial detergents. Cio-sucrose Assay for Antitumor Activity in Vivo. Antitumor activity dz-FA was assayed with the use of Ehrlich ascites tumor in mice. Ci2-Me ester About 1 million of the tumor cells were implanted i.p. in ddY mice, 5 weeks old, weighing 18 to 22 g. A solution or suspension of the samples in 0.86% NaCl solution was Agents without antitumor activity administered once daily for 5 successive days, and the tumor Tween 20 growth and body weight gain after 7 days and life-span up to Tween 40 30 days were observed. The life-span was shown as the average life-span of 10 mice in each group. I 2345678 Assay for Hemolytic Activity. Hemolytic activity of the Chart 1. Hemolytic activity of fatty acid esters. Samples were esters of fatty acids was assayed with the use of sheep red dissolved or suspended in 0.86% NaCl solution at serial dilutions blood cells. Sheep red blood cells were collected from the starting with 1 mg/ml, and an equal volume of the sheep red blood cell commercial sheep red blood cell suspension (Toshiba Kagaku, suspension in the NaCl solution was added. After the incubation at 37Â° Tokyo, Japan) by centrifugation at 1500 rpm for 5 min, for 4 hr, the hemolytic activity was determined. The activity was washed with 0.86% NaCl solution 3 times, and suspended in expressed by the highest dilution in which hemolysis was observed. PC, 100 volumes of the NaCl solution. Samples were dissolved or propylene glycol; AfC, monoglyceride;FA, fatty acid;A/e, methyl. suspended in the 0.86% NaCl solution at the serial dilutions, and an equal volume of the red cell suspension was added. Assay for Viability of Tumor Cells. Ascitic tumor cells of After incubation at 37Â°,for 4 hr, the hemolytic activity was Ehrlich carcinoma maintained by weekly i.p. transplantation were washed 3 times with PBS1 and suspended at a determined. The activity was expressed by the highest dilution in which hemolysis was observed visually. 1The abbreviation used is: PBS, phosphate-buffered saline.

502 CANCER RESEARCH VOL. 31

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1971 American Association for Cancer Research. An titumor Activity of Some Fatty Acids concentration of 1 X IO6 cells/ml. Equal volumes of the the mixture was determined by counting the number of sample in PBS and the tumor cell suspension were mixed and cells microscopically. incubated at 37Â°for 1 hr. The viability of the treated tumor Assay for Transplantability of Tumor Cells. Ehrlich ascites cells was determined by staining the cells with safranin dye tumor cells were treated as described for assay for viability according to the method described by Oda (14). To the except that the tumor cells were suspended at a concentration of 1 X IO7 cells/ml. After the incubation at 37Â°for 1hr, 0.2 reaction mixture, safranin solution in PBS was added to a final concentration of 0.05%. The percentage of the dead cells in ml of the mixture was implanted i.p. into a ddY mouse. The survival time and tumor growth of the each mouse were ob Table 2 served. Ten mice were used for each group, and the control Effect of some fatty acid esters on Ehrlich ascites tumor cells mice were given injections of untreated tumor cells. Figures indicate the percentage of cells stained with safranin. To 0.5 ml of the tumor cell suspension (10* cells/ml), 0.5 ml of the sample suspension or solution was added. After incubation at 37Â°for 1 hr, RESULTS safranin was added at a final concentration of 0.05%. The percentage of dead cells was determined by counting the stained cells microscopically. C, â€ž,C,,, C14, C,0,, and C,28>indicate the esters of capric, lauric, In vivo antitumor activity of some of the fatty acid esters is myristic, stearic, and linoleic acids, respectively. shown in Table 1. While monolaurin and sucrose monolaurate show activity, sorbitan laurate and polyoxyethylene sorbitan Percent at monolaurate (Tween 20) have no activity. Studies on the 500 50 5 activity of other esters (8) showed that most of the sucrose Compounds Mg/ml Mg/ml Mg/ml esters of fatty acids and the propylene glycol ester of myristic acid were the most effective against the tumor tested and that AgentsactivityC, with strong antitumor -,sucroseC, Tweens, sorbitan esters, and most of the methyl esters were esterFungal4 propylene glycol ineffective. monoglycerideAgents 3010052891003 Hemolytic activity of some of the fatty acid esters was activityC,with weak antitumor studied by the method described in "Materials and Methods." jmonoglycerideC, The purpose of this experiment was to examine whether there â€žsucroseC, esterAgents2 methyl would exist a parallel relationship between hemolytic activity activityC,Â°8without antitumor and in vivo antitumor activity. The result is presented in Chart monoglycerideC,% 1. Hemolytic activity was expressed by the highest dilution in methylesterTween which hemolysis was observed. As can be seen from the chart, 20Control100100100100994310010063100 some of the esters with strong in vivo activity show strong hemolytic activity while others show very weak activity.

Table 3 Viability and transportability of tumor cells treated by some esters at 2500 or 500 ng/W cells Equal volumes of tumor cell suspension (2 X IO7 cells/ml) and the sample suspension or solution (5 mg/ml or 1 mg/ml) were mixed and incubated at 37Â°for 1 ht. Viability was determined by staining the cells with safranin, and transplantability was determined by implanting 0.2 ml of the incubated mixture into a ddY mouse. Ten mice were used for each group. The figures after the compounds indicate the final concentration of the compound in mg/107 cells.

without cells(%)980880940850670870580102152Micetumor after time CompoundsNoneLauric 7 days(%)0100010001000100010001000100808040Survival(days)18.8>30.020.8>29.920.1>28.422.0>30.020.8>29.319.2>30.021.6>30.0>29.0>29.4>27.0

esterPalmiticsucrose

esterOleicsucrose

esterLinoleicsucrose

esterElaidicsucrose

esterPropylenesucrose

myristateTweenglycol

20Tween

802.50.52.50.52.50.52.50.52.50.52.50.52.50.52.50.5Viable

MAY 1971 503

Tween 20, which showed no in vivo activity, demonstrated Activity from Fungal Mycelia. J. Antibiotics (Tokyo), 22: 18-22, strong hemolytic activity. Thus there is no relationship 1969. between antitumor activity and hemolytic activity. 4. Bennett, L. R., and Connon, F. E. Effect of Lytic Agents on Next, we examined in vitro action of those lipids on the Plasma Membrane of Ehrlich Ascites Tumor Cells and Mouse tumor cells, thinking that each lipid might have specific Erythrocytes. J. Nati. Cancer Inst., 19: 999-1011, 1957. affinity to the tumor cells and that those with strong affinity 5. Kato, A., Ando, K., Kodama, K., Suzuki, S., Suzuki, K., Tamura, attack the cell and show antitumor activity. In vitro attack of G., and Arima, K. Production, Isolation and Purification of Antitumor Active Monoglycerides and Other Antibiotics from the cell was measured by viability and transplantability of the treated tumor cells. The method is described in "Materials and Sepedonium ampullosporum. J. Antibiotics (Tokyo), 22: 67-72, Methods." In Table 2, viability of the tumor cells treated with 1969. 6. Kato, A., Ando, K., Kodama, K., Tamura, G., and Arima, K. some esters at 3 doses is presented with the reference to their Identification and Chemical Properties of Antitumor Active antitumor activity. Here again, we see no strict relationship Monoglycerides from Fungal Mycelia. J. Antibiotics (Tokyo), 22: between in vitro and in vivo activities. Table 3 shows the 73-78, 1969. viability and transplantability of the tumor cells treated with 7. Kato, A., Ando, K., Suzuki, S., Tamura, G., and Arima, K. sucrose and propylene glycol esters of fatty acids and Tweens Antitumor Activity of Monoglycerides and Other Esters of Fatty at concentrations of 2500 and 500 Mg/107 cells. At the higher Acids. J. Antibiotics (Tokyo), 22: 83-84, 1969. concentration, none of the treated cells were viable; when 8. Kato, A., Ando, K., Suzuki, S., Tamura, G., and Arima, K. Antitumor Activity of Fatty Acids and Their Esters. II. Activity of inoculated into mice, these cells did not produce tumors. When the concentration was reduced to 500/ug/107 cells, none Monoglycerides and Other Esters. In: H. Umezawa (ed.), Progress in Antimicrobial and Anticancer Chemotherapy, Proceedings of the of the sucrose esters and propylene glycol ester showed any Sixth International Congress of Chemotherapy, Vol. 2, p. 142. cytotoxic effects on the tumor cells. However, 90% of the cells Tokyo, Japan: University of Tokyo Press, 1970. treated with Tween 20 at the same concentration, 500/ug/107 9. Morgan, J. F., Tolnai, S., and Townsend, G. F. Studies on the in cells, lost their viability and consequently their Vitro Antitumor Activity of Fatty Â»-Acids. II. Saturated transplantability. Dicarboxylic Acids. Can. J. Biochem. Physiol., 38: 597-603, 1960. 10. Nakahara, W. Studies on Lymphoid Activity. VI. Immunity to DISCUSSION Transplanted Cancer Induced by Injection of Olive Oil. J. Exptl. Med., 35: 493-507, 1922. It is significant that fatty acid esters with strong in vivo 11. Nakahara, W. Effect of Fatty Acids on the Resistance of Mice to activity were less effective in vitro than Tween 20, which did Transplanted Cancer. J. Exptl. Med., 40: 363-373, 1924. 12. Nakahara, W. Resistance to Spontaneous Mouse Cancer Induced by not show antitumor activity in vivo. Thus antitumor activity Injections of Oleic Acid. J. Exptl. Med., 41: 347-357,1925. shown by sucrose monoesters and some other esters of fatty 13. Nakahara, W. Production of Tumor Resistance by Fatty Acids. acids cannot be explained only by their physical attack of the Gann, 19: 1-4, 1925. tumor cells. Differences in physiological and biochemical 14. Oda, A. Differential Staining Method for Dead and Live Tumor properties of the esters should differentiate their antitumor Cells. Japan. J. Exptl. Med., 29: 79-86, 1959. activity. Further studies are under way. 15. Osipow, L., Snell, F. D., York, W. C, and Finchler, A. Fatty Acid This is the first report in which sucrose esters of fatty acids Esters of Scurose. Ind. Eng. Chem., 48: 1459-1462, 1956. are used in biological studies. Our results suggest the 16. Seno, S., and Yamamoto, M. Chemical Analysis and Biological possibility that sucrose esters of fatty acids could be used as Activities of Fatty Acids from the Liver of X-ray Irradiated Rabbit, water-soluble derivatives of fatty acids in many experiments. the Antitumor Agent So-called OX. Acta Med. Okayama, 19: 59-72, 1965. 17. Tolnai, S., and Morgan, J. F. Studies on the in Vitro Antitumor ACKNOWLEDGMENTS Activity of Fatty Acids. III. Saturated Monocarboxylic Acids. Can. J. Biochem. Physiol., 39: 713-719, 1961. We are thankful to Mr. Seikichi Suzuki for his kind advice and 18. Tolnai, S., and Morgan, J. F. Studies on the in Vitro Antitumor discussions. Activity of Fatty Acids. V. Unsaturated Acids. Can. J. Biochem. Physiol., 40: 869-875, 1962. 19. Tolnai, S., and Morgan J. F. Studies on the in Vitro Antitumor REFERENCES Activity of Fatty Acids. VI. Derivatives of Mono- and Di-carboxylic and Unsaturated Fatty Acids. Can. J. Biochem. 1. Ando, K., Kalo, A., Suzuki, S., Tamura, G., and Arima, K. Physiol., 40: 1367-1373, 1962. Antitumor Activity of Fatty Acids and Their Esters. I. Activity of 20. Tolnai, S., and Morgan, J. F. Studies on the in Vitro Antitumor Fatty Acids. In: H. Umezawa (ed.), Progress in Antimicrobial and Activity of Fatty Acids. VII. Effect of Amino Acid-Fatty Acid Anticancer Chemotherapy, Proceedings of the Sixth International Salts. Can. J. Biochem., 44: 979-981, 1966. Congress of Chemotherapy, Vol. 2, p. 136. Tokyo, Japan: 21. Townsend, G. F., Morgan, J. F., and Hazlett, B. Activity of University of Tokyo Press, 1970. 10-Hydroxydecenoic Acid from Royal Jelly against Experimental 2. Ando, K., Kato, A., Tamura, G., and Arima, K. Chemical Study of Leukemia and Ascitic Tumors. Nature, 183: 1270-1271, 1959. Fatty Acids with Antitumor Activity Isolated from Fungal 22. Townsend, G. F., Morgan, J. F., Tolnai, S., Hazlett, B., Morton, H. Mycelia. J. Antibiotics (Tokyo), 22: 23-26, 1969. J., and Shuel, R. W. Studies on the in Vitro Antitumor Activity of 3. Ando, K., Suzuki, S., Suzuki, K., Kodama, K., Kato, A., Tamura, Fatty Acids. I. lO-Hydroxy-2-decenoic Acid from Royal Jelly. G., and Arima, K. Isolation of Fatty Acids with Antitumor Cancer Res., 20: 503-510, 1960.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1971 American Association for Cancer Research. Effects of Some Fatty Acid Esters on the Viability and Transplantability of Ehrlich Ascites Tumor Cells

Akiko Kato, Kunio Ando, Gakuzo Tamura, et al.

Cancer Res 1971;31:501-504.

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